The objective of this proposal is to develop a general methodology for the rapid identification of intersubunit interfaces in protein complexes, and utilize it to identify the subunit/subunit interfaces in immature and mature HIV-1 capsids, The strategy will be to synthesize a family of biotin-tagged chemical cross-linkers with both specific and non-specific cross-linking activity and use them as distance constraints to position subunits of known atomic structure (in this case the structural proteins of HIV) relative to one another in space, To achieve the necessary structural resolution the cross-linked amino acid residue pairs will be identified by mass spectrometry. This project extends the parent grant (AI44626) whose mission is to utilize hydrogen/deuterium exchange studies to identify intersubunit interfaces and dynamic motions in HIV capsids. The novel component of this proposal is the synthesis of biotin tagged cross-linkers, which can be rapidly purified using strepavidin affinity techniques coupled with mass spectrometry for identification of the cross-linked peptides. Such cross-linkers are not commercially available. Our preliminary data indicates that while the cross-linking data can be used to pack subunits, its widespread applicability is limited by the inherent difficulty of detecting a small number of cross-linked peptides against a background of a large number of uncross-linked peptides. Biotin tagged cross-linkers can be rapidly purified from the complex digests and analyzed by mass spectrometry resulting in increases in sensitivity and throughput. As a result the technique will become a generally applicable tool for merging data from the structural genomics and proteomics initiatives.